Naphthyl compounds, intermediates, compositions, and methods

ABSTRACT

The present invention is related to methods for treating pathological conditions resulting from an estrogen deficiency, such as cardiovascular disease and osteoporosis, and is further related to methods for treating estrogen-dependent cancer, such as breast and uterine cancer, employing compounds of formula I

This application is a division of prior application Ser. No. 08/395,950,filed on Feb. 28, 1995 now abandoned.

FIELD OF THE INVENTION

This invention relates to the fields of pharmaceutical and organicchemistry and provides novel naphthyl compounds which are useful for thetreatment of the various medical indications associated withpost-menopausal syndrome, and uterine fibroid disease, endometriosis,and aortal smooth muscle cell proliferation. The present inventionfurther relates to intermediate compounds useful for preparing thepharmaceutically active compounds of the present invention, andpharmaceutical compositions.

BACKGROUND OF THE INVENTION

“Post-menopausal syndrome” is a term used to describe variouspathological conditions which frequently affect women who have enteredinto or completed the physiological metamorphosis known as menopause.Although numerous pathologies are contemplated by the use of this term,three major effects of post-menopausal syndrome are the source of thegreatest long-term medical concern: osteoporosis, cardiovascular effectssuch as hyperlipidemia, and estrogen-dependent cancer, particularlybreast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverseetiologies, but which are characterized by the net loss of bone mass perunit volume. The consequence of this loss of bone mass and resultingbone fracture is the failure of the skeleton to provide adequatestructural support for the body. One of the most common types ofosteoporosis is that associated with menopause. Most women lose fromabout 20% to about 60% of the bone mass in the trabecular compartment ofthe bone within 3 to 6 years after the cessation of menses. This rapidloss is generally associated with an increase of bone resorption andformation. However, the resorptive cycle is more dominant and the resultis a net loss of bone mass. Osteoporosis is a common and serious diseaseamong post-menopausal women.

There are an estimated 25 million women in the United States, alone, whoare afflicted with this disease. The results of osteoporosis arepersonally harmful and also account for a large economic loss due itschronicity and the need for extensive and long term support(hospitalization and nursing home care) from the disease sequelae. Thisis especially true in more elderly patients. Additionally, althoughosteoporosis is not generally thought of as a life threateningcondition, a 20% to 30% mortality rate is related with hip fractures inelderly women. A large percentage of this mortality rate can be directlyassociated with post-menopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausalosteoporosis is the trabecular bone. This tissue is often referred to asspongy or cancellous bone and is particularly concentrated near the endsof the bone (near the joints) and in the vertebrae of the spine. Thetrabecular tissue is characterized by small osteoid structures whichinter-connect with each other, as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This inter-connected network of trabeculae gives lateralsupport to the outer cortical structure and is critical to thebio-mechanical strength of the overall structure. In post-menopausalosteoporosis, it is, primarily, the net resorption and loss of thetrabeculae which leads to the failure and fracture of bone. In light ofthe loss of the trabeculae in post-menopausal women, it is notsurprising that the most common fractures are those associated withbones which are highly dependent on trabecular support, e.g., thevertebrae, the neck of the weight bearing bones such as the femur andthe fore-arm. Indeed, hip fracture, collies fractures, and vertebralcrush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the only generally accepted method for treatment ofpost-menopausal osteoporosis is estrogen replacement therapy. Althoughtherapy is generally successful, patient compliance with the therapy islow primarily because estrogen treatment frequently produces undesirableside effects.

Throughout premenopausal time, most women have less incidence ofcardiovascular disease than age-matched men. Following menopause,however, the rate of cardiovascular disease in women slowly increases tomatch the rate seen in men. This loss of protection has been linked tothe loss of estrogen and, in particular, to the loss of estrogen'sability to regulate the levels of serum lipids. The nature of estrogen'sability to regulate serum lipids is not well understood, but evidence todate indicates that estrogen can upregulate the low density lipid (LDL)receptors in the liver to remove excess cholesterol. Additionally,estrogen appears to have some effect on the biosynthesis of cholesterol,and other beneficial effects on cardiovascular health.

It has been reported in the literature that post-menopausal women havingestrogen replacement therapy have a return of serum lipid levels toconcentrations to those of the pre-menopausal state. Thus, estrogenwould appear to be a reasonable treatment for this condition. However,the side-effects of estrogen replacement therapy are not acceptable tomany women, thus limiting the use of this therapy. An ideal therapy forthis condition would be an agent which would regulate the serum lipidlevel as does estrogen, but would be devoid of the side-effects andrisks associated with estrogen therapy.

The third major pathology associated with post-menopausal syndrome isestrogen-dependent breast cancer and, to a lesser extent,estrogen-dependent cancers of other organs, particularly the uterus.Although such neoplasms are not solely limited to a post-menopausalwomen, they are more prevalent in the older, post-menopausal population.Current chemotherapy of these cancers has relied heavily on the use ofanti-estrogen compounds such as, for example, tamoxifen. Although suchmixed agonist-antagonists have beneficial effects in the treatment ofthese cancers, and the estrogenic side-effects are tolerable in acutelife-threatening situations, they are not ideal. For example, theseagents may have stimulatory effects on certain cancer cell populationsin the uterus due to their estrogenic (agonist) properties and they may,therefore, be contraproductive in some cases. A better therapy for thetreatment of these cancers would be an agent which is an anti-estrogencompound having negligible or no estrogen agonist properties onreproductive tissues.

In response to the clear need for new pharmaceutical agents which arecapable of alleviating the symptoms of, inter alia, post-menopausalsyndrome, the present invention provides new benzothiophene compounds,pharmaceutical compositions thereof, and methods of using such compoundsfor the treatment of post-menopausal syndrome and other estrogen-relatedpathological conditions such as those mentioned below.

Uterine fibrosis (uterine fibroid disease) is an old and ever presentclinical problem which goes under a variety of names, including uterinefibroid disease, uterine hypertrophy, uterine lieomyomata, myometrialhypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterinefibrosis is a condition where there is an inappropriate deposition offibroid tissue on the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibrosis involves surgicalprocedures both costly and sometimes a source of complications such asthe formation of abdominal adhesions and infections. In some patients,initial surgery is only a temporary treatment and the fibroids regrow.In those cases a hysterectomy is performed which effectively ends thefibroids but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, yettheir use is tempered by the fact they can lead to osteoporosis. Thus,there exists a need for new methods for treating uterine fibrosis, andthe methods of the present invention satisfy that need.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release and subsequent ovarian production ofestrogen; however, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinswhich induces amenorrhea and by suppressing ovarian estrogen productioncan cause regressions of the endometrial growths. The use of chronicprogestin therapy is often accompanied by the unpleasant CNS sideeffects of progestins and often leads to infertility due to suppressionof ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis; however, theyinduce severe masculinizing effects. Several of these treatments forendometriosis have also been implicated in causing a mild degree of boneloss with continued therapy. Therefore, new methods of treatingendometriosis are desirable.

Smooth aortal muscle cell proliferation plays an important role indiseases such as atherosclerosis and restenosis. Vascular restenosisafter percutaneous transluminal coronary angioplasty (PTCA) has beenshown to be a tissue response characterized by an early and late phase.The early phase occurring hours to days after PTCA is due to thrombosiswith some vasospasms while the late phase appears to be dominated byexcessive proliferation and migration of aortal smooth muscle cells. Inthis disease, the increased cell motility and colonization by suchmuscle cells and macrophages contribute significantly to thepathogenesis of the disease. The excessive proliferation and migrationof vascular aortal smooth muscle cells may be the primary mechanism tothe reocclusion of coronary arteries following PTCA, atherectomy, laserangioplasty and arterial bypass graft surgery. See “IntimalProliferation of Smooth Muscle Cells as an Explanation for RecurrentCoronary Artery Stenosis after Percutaneous Transluminal CoronaryAngioplasty,” Austin et al., Journal of the American College ofCardiology, 8: 369-375 (August 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty and arterialbypass graft surgery. In about 35% of the patients who undergo PTCA,reocclusion occurs within three to six months after the procedure. Thecurrent strategies for treating vascular restenosis include mechanicalintervention by devices such as stents or pharmacologic therapiesincluding heparin, low molecular weight heparin, coumarin, aspirin, fishoil, calcium antagonist, steroids, and prostacyclin. These strategieshave failed to curb the reocclusion rate and have been ineffective forthe treatment and prevention of vascular restenosis. See “Prevention ofRestenosis after Percutaneous Transluminal Coronary Angioplasty: TheSearch for a ‘Magic Bullet’,” Hermans et al., American Heart Journal,122: 171-187 (July 1991).

In the pathogenesis of restenosis excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall whichmediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth aortalmuscle cells are useful in the treatment and prevention of restenosis.The present invention provides for the use of compounds as smooth aortalmuscle cell proliferation inhibitors and, thus inhibitors of restenosis.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formula I

wherein

R¹ is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), or—OSO₂(C₂-C₆ alkyl);

R² is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), —OSO₂(C₂-C₆alkyl), or halo;

R³ is 1-piperidinyl, 1-pyrrolidinyl, methyl-1-pyrrolidinyl,dimethyl-1-pyrrolidino, 4-morpholino, dimethylamino, diethylamino,diisopropylamino, or 1-hexamethyleneimino; and

n is 2 or 3; and or a pharmaceutically acceptable salt thereof.

Also provided by the present invention are intermediate compounds offormula II which are useful for preparing the pharmaceutically activecompounds of the present invention, and are shown below

wherein

R^(1a) is —H or —OR⁵ in which R⁵ is a hydroxy protecting group.

R^(2a) is —H, halo, or —OR⁶ in which R⁶ is a hydroxy protecting group;and

R⁴ is —OH or —CHO; or a pharmaceutically acceptable salt thereof.

The present invention further relates to pharmaceutical compositionscontaining compounds of formula I, optionally containing estrogen orprogestin, and the use of such compounds, alone, or in combination withestrogen or progestin, for alleviating the symptoms of post-menopausalsyndrome, particularly osteoporosis, cardiovascular related pathologicalconditions, and estrogen-dependent cancer. As used herein, the term“estrogen” includes steroidal compounds having estrogenic activity suchas, for example, 17β-estradiol, estrone, conjugated estrogen(Premarin®), equine estrogen 17β-ethynyl estradiol, and the like. Asused herein, the term “progestin” includes compounds havingprogestational activity such as, for example, progesterone,norethylnodrel, nongestrel, megestrol acetate, norethindrone, and thelike.

The compounds of the present invention also are useful for inhibitinguterine fibroid disease and endometriosis in women, and aortal smoothmuscle cell proliferation, particularly restenosis, in humans.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention includes compounds of formula I

wherein

R¹ is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), or—OSO₂(C₂-C₆ alkyl);

R² is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), —OSO₂(C₂-C₆alkyl), or halo;

R³ is 1-piperidinyl, 1-pyrrolidinyl, methyl-1-pyrrolidinyl,dimethyl-1-pyrrolidino, 4-morpholino, dimethylamino, diethylamino,diisopropylamino, or 1-hexamethyleneimino; and

n is 2 or 3; and or a pharmaceutically acceptable salt thereof.

General terms used in the description of compounds herein described beartheir usual meanings. For example, “C₁-C₆ alkyl” refers to straight orbranched aliphatic chains of 1 to 6 carbon atoms including moieties suchas methyl, ethyl, propyl, isopropyl, butyl, n-butyl, pentyl, isopentyl,hexyl, isohexyl, and the like. Similarly, the term “C₁-C₄ alkoxy”represents a C₁-C₄ alkyl group attached through an oxygen molecule andinclude moieties such as, for example, methoxy, ethoxy, n-propoxy,isopropoxy, and the like. Of these alkoxy groups, methoxy is highlypreferred in most circumstances.

The starting material for preparing compounds of the present inventionis a compound of formula III

wherein

R^(1a) is —H or —OR⁵ in which R⁵ is a hydroxy protecting group; and

R^(2a) is —H, halo, or —OR⁶ in which R⁶ is a hydroxy protecting group.Compounds of formula III are well known in the art and are preparedessentially as described by Boyle, et al., in U.S. Pat. No. 4,910,212which is herein incorporated by reference. See., also, Collins, D. J.,et al., Aust. J. Chem., 41: 745-756 (1988); and Collins, D. J., et al.,Aust. J. Chem., 37: 2279-2294 (1984).

In preparing compounds of the present invention, generally, a ketone offormula III is aromatized, providing a phenol of formula IV, which isthen reacted with a 4-halobenzaldehyde to give a biaryl ether of formulaIIa, which, in turn, is converted to a phenol of formula IIb. Thissynthetic route is as shown below in Scheme I, and R^(1a) and R^(2a) areas defined above.

In the first step of the present process, a compound of formula III isconverted to a phenol of formula IV via a three-step protocol,essentially as described by Wang, G., et al., M. Syn. Commun., 21: 989(1991). In essence, a formula II ketone is enolized by refluxing asolution of a formula III compound in an appropriate acetate solvent, inthe presence of an acid catalyst. The resulting enolacetate is directlyconverted to a naphtholacetate which is then hydrolyzed to a phenol offormula IV.

In converting a ketone of formula III to its respective enol, variousknown acid catalysts can be used. Preferably, non-aqueous acids, andparticularly, p-toluenesulfonic acid is preferred.

Appropriate acetate solvents include, for example, simple alcohol estersof acetate acid, particularly isopropenyl acetate.

When run at reflux, the present reaction takes from about 6 to about 48hours to complete. The enol product from this reaction is not isolated,but upon completion of the reaction, the resulting solution is treatedwith an appropriate oxidant and heated to reflux for, optimally, about 1to about 3 hours.

Appropriate oxidants for this second phase of the first reaction stepshown in Scheme I are limited to those known in the art which can leadto the elimination of a hydrogen atom from a saturated system to give anaromatized system. Such oxidants include, for example, hydrogenationcatalysts such as platinum, palladium, and nickel, elemental sulfur andselenium, and quinones. For the present application, quinone oxidants,especially 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) arepreferred. About 1 to 2 equivalents of DDQ per equivalent of substratewill drive the present process phase.

The resulting product of the present phase, a naphtholacetate, is thensubjected to hydrolysis to provide a compound of formula IV, thuscompleting the first process step shown in Scheme I. The presenthydrolysis phase is accomplished via either acid or basic hydrolysis ofthe substrate in a polar protic solvent such as water or one or moresolvents containing an alcohol such as methanol or ethanol. A cosolventsuch as tetrahydrofuran (THF) or dioxane also may be added to thesolution to aid solubility. Appropriate bases for this phase includesodium hydroxide, potassium hydroxide, lithium hydroxide, and the like.Appropriate acids include, for example, hydrochloric acid,methanesulfonic acid, p-toluenesulfonic acid, and the like.

This final phase of the first step shown in Scheme I, supra, can be runat ambient temperature and runs in a short period of time, typicallyfrom 1 to about 12 hours. Completion of the present reaction can bedetermined via standard chromatographic techniques such as thin layerchromatography.

In the second step of Scheme I, a phenol of formula IV is first reactedwith a base, followed by the addition of a 4-halobenzaldehyde in a polaraprotic solvent, under an inert atmosphere such as nitrogen, to give abiarylether of formula IIa. This reaction is well known in the art andis carried out essentially as described by Yeager, G. W., et al.,Synthesis, 63 (1991).

More particularly, 1 equivalent of a formula IV compound is firsttreated with at least 1 equivalent of an alkali metal hydride orcarbonate in an appropriate solvent, followed by a dropwise addition ofa 4-halobenzaldehyde in the same solvent as used with the substrate.

Appropriate solvents for this reaction are those solvents or mixture ofsolvents which remain inert throughout the reaction.N,N-dimethylformamide (DMF), especially the anhydrous form thereof, ispreferred.

Preferably, sodium hydride is used as the required base, and4-fluorobenzaldehyde is used as the preferred 4-halobenzaldehyde.

The temperature employed in this step of the present process should besufficient to effect completion of this reaction, without encouragingthe formation of undesirable by-products. A preferred temperature rangefor this reaction is from about 30° C. to about 100° C.

Under preferred reaction conditions, a formula IIa compound will beprepared via the preferred process in about 24 to about 48 hours.

The final reaction shown in Scheme I, the conversion of the aldehydemoiety of a formula IIa compound to a phenol group, thus forming acompound of formula IIb, is known in the art as a Bayer-Villigeroxidation. See, e g., Fiesers, L., et al., Reagents for OrganicSynthesis, 1: 467, Wiley (New York, 1967); Hassall, C. H., OrganicReactions, 9: 73-106 (Wiley, N.Y., 1967).

In general, the present reaction involves the combination of abenzaldehyde with a peracid such as peracetic acid or m-chloroperbenzoicacid in an inert solvent such as chloroform or methylenechloride. Theproduct of this reaction, a formate ester, can then be readilyhydrolyzed to the desired phenol. See, e.g., Yeager, et al., supra;Godfrey, I. M., et al., J. Chem. Soc. Perkins. Trans. I:1353 (1974); andRue, R., et al., Bull. Soc. Shim. Fr., 3617 (1970).

For the present reaction, a preferred variation is described byMatsumoto, M., et al., J. Org. Chem., 49: 4741 (1984). This methodinvolves combining a benzaldehyde of formula IIa with at least 1 toabout 2 equivalents of 30% hydrogen peroxide in an alcohol solvent, andin the presence of a catalytic acid. Under these conditions, the phenolis formed directly, and the need for an additional hydrolysis step is,therefore, eliminated.

The preferred solvent and acid catalyst for the present reaction ismethanol and concentrated sulfuric acid, respectively.

Under the preferred reaction conditions, the transformation from aformula IIa compound to a formula IIb compound is complete afterstirring for about 12 to about 48 hours at ambient temperatures.

Compounds of formula IIa and IIb collectively are herein depicted asnovel intermediate compounds of formula II which are useful for thepreparation of pharmaceutically active compounds of formula I of thepresent invention.

Upon preparation of a formula IIb compound, it is reacted with acompound of formula V

R³—(CH₂)_(n)—Q  V

wherein R³ and n are as defined above and Q is a bromo or, preferably, achloro moiety, to form a compound of formula Ia. The formula Ia compoundis then deprotected, when R⁵ and/or R⁶ hydroxy protecting groups arepresent, to form a compound of formula Ib. These process steps are shownin Scheme II below.

wherein

R^(1a), R^(2a), R³, and n are as defined above;

R^(1b) is —H or —OH; and

R^(2b) is —H, —OH, or halo; or a pharmaceutically acceptable saltthereof.

In the first step of the process shown in Scheme II, the alkylation iscarried out via standard procedures. Compounds of formula V arecommercially available or are prepared by means well known to one ofordinary skill in the art. Preferably, the hydrochloride salt of aformula V compound, particularly 2-chloroethylpiperidine hydrochloride,is used.

Generally, at least about 1 equivalent of formula IIb substrate arereacted with 2 equivalents of a formula V compound in the presence of atleast about 4 equivalents of an alkali metal carbonate, preferablycesium carbonate, and an appropriate solvent.

Solvents for this reaction are those solvents or mixture of solventswhich remain inert throughout the reaction. N,N-dimethylformamide,especially the anhydrous form thereof, is preferred.

The temperature employed in this step should be sufficient to effectcompletion of this alkylation reaction. Typically, ambient temperatureis sufficient and preferred.

The present reaction preferably is run under an inert atmosphere,particularly nitrogen.

Under the preferred reaction conditions, this reaction will run tocompletion in about 16 to about 20 hours. Of course, the progress of thereaction can be monitored via standard chromatographic techniques.

As an alternative for preparing compounds of formula Ia, a formula IIbcompound is reacted with an excess of an alkylating agent of the formula

Q—(CH₂)_(n)—Q′

wherein Q and Q′ each are the same or different leaving group, in analkali solution. Appropriate leaving groups include the sulfonates suchas methanesulfonate, 4-bromobenzenesulfonate, toluenesulfonate,ethanesulfonate, isopropylsulfonate, 4-methoxybenzenesulfonate,4-nitrobenzenesulfonate, 2-chlorobenzenesulfonate, triflate, and thelike, halogens such as bromo, chloro, and iodo, and other relatedleaving groups. Aulogens are preferred leaving groups and bromo isespecially preferred.

A preferred alkali solution for this alkylation reaction containspotassium carbonate in an inert solvent such as, for example, methyethylketone (MEK) or DMF. In this solution, the 4-hydroxy group of thebenzoyl moiety of a formula IIb compound exists as a phenoxide ion whichdisplaces one of the leaving groups of the alkylating agent.

This reaction is best when the alkali solution containing the reactantsand reagents is brought to reflux and allowed to run to completion. Whenusing MEK as the preferred solvent, reaction times run from about 6hours to about 20 hours.

The reaction product from this step is then reacted with 1-piperidine,1-pyrrolidine, methyl-1-pyrrolidine, dimethyl-1-pyrrolidine,4-morpholine, dimethylamine, diethylamine, or 1-hexamethyleneimine, viastandard techniques, to form compounds of formula Ia. Preferably, thehydrochloride salt of piperidine is reacted with the alkylated compoundof formula IIb in an inert solvent, such as anhydrous DMF, and heated toa temperature in the range from about 60° C. to about 110° C. When themixture is heated to a preferred temperature of about 90° C., thereaction only takes about 30 minutes to about 1 hour. However, changesin the reaction conditions will influence the amount of time thisreaction needs to be run to completion. Of course, the progress of thisreaction step can be monitored via standard chromatographic techniques.

Compounds of formula Ia, in which R⁵ and/or R⁶, when present, are C₁-C₄alkyl, preferably methyl, are novel and are pharmaceutically active forthe methods herein described. Accordingly, such compounds areencompassed by the definition herein of compounds of formula I.

Preferred compounds of formula I are obtained by cleaving, when present,the R⁵ and R⁶ hydroxy protecting groups of formula Ia compounds via wellknown procedures. Numerous reactions for the formation and removal ofsuch protecting groups are described in a number of standard worksincluding, for example, Protective Groups in Organic Chemistry, PlenumPress (London and New York, 1973); Green, T. W., Protective Groups inOrganic Synthesis, Wiley, (New York, 1981); and The Peptides, Vol. I,Schrooder and Lubke, Academic Press (London and New York, 1965). Methodsfor removing preferred R⁵ and/or R⁶ hydroxy protecting groups,particularly methyl, are essentially as described in Example 5, infra.

Compounds of formula Ia are novel, are pharmaceutically active for themethods herein described, and are encompassed by formula I as definedherein.

Other preferred compounds of formula I are prepared by replacing the 6-and/or 4′-position hydroxy moieties, when present, with a moiety of theformula —O—CO—(C₁-C₆ alkyl), or —O—SO₂—(C₂-C₆ alkyl) via well knownprocedures. See, e.g., U.S. Pat. No. 4,358,593.

For example, when an —O—CO(C₁-C₆ alkyl) group is desired, a mono- ordihydroxy compound of formula I is reacted with an agent such as acylchloride, bromide, cyanide, or azide, or with an appropriate anhydrideor mixed anhydride. The reactions are conveniently carried out in abasic solvent such as pyridine, lutidine, quinoline or isoquinoline, orin a tertiary amine solvent such as triethylamine, tributylamine,methylpiperidine, and the like. The reaction also may be carried out inan inert solvent such as ethyl acetate, dimethylformamide,dimethylsulfoxide, dioxane, dimethoxyethane, acetonitrile, acetone,methyl ethyl ketone, and the like, to which at least one equivalent ofan acid scavenger (except as noted below), such as a tertiary amine, hasbeen added. If desired, acylation catalysts such as4-dimethylaminopyridine or 4-pyrrolidinopyridine may be used. See, e.g.,Haslam, et al., Tetrahedron, 36: 2409-2433 (1980).

The present reactions are carried out at moderate temperatures, in therange from about −25° C. to about 100° C., frequently under an inertatmosphere such as nitrogen gas. However, ambient temperature is usuallyadequate for the reaction to run.

Acylation of a 6-position and/or 4′-position hydroxy group also may beperformed by acid-catalyzed reactions of the appropriate carboxylicacids in inert organic solvents. Acid catalysts such as sulfuric acid,polyphosphoric acid, methanesulfonic acid, and the like are used.

The aforementioned R¹ and/or R² groups of formula I compounds also maybe provided by forming an active ester of the appropriate acid, such asthe esters formed by such known reagents such asdicyclohexylcarbodiimide, acylimidazoles, nitrophenols,pentachlorophenol, N-hydroxysuccinimide, and 1-hydroxybenzotriazole.See, e.g., Bull. Chem. Soc. Japan, 38: 1979 (1965), and Chem. Ber., 788and 2024 (1970).

Each of the above techniques which provide —O—CO—(C₁-C₆ alkyl) moietiesare carried out in solvents as discussed above. Those techniques whichdo not produce an acid product in the course of the reaction, of course,do not call for the use of an acid scavenger in the reaction mixture.

When a formula I compound is desired in which the 6-and/or 4′-positionhydroxy group of a formula I compound is converted to a group of theformula —O—SO₂—(C₂-C₆ alkyl), the mono- or dihydroxy compound is reactedwith, for example, a sulfonic anhydride or a derivative of theappropriate sulfonic acid such as a sulfonyl chloride, bromide, orsulfonyl ammonium salt, as taught by King and Monoir, J. Am. Chem. Soc.,97: 2566-2567 (1975). The dihydroxy compound also can be reacted withthe appropriate sulfonic anhydride or mixed sulfonic anhydrides. Suchreactions are carried out under conditions such as were explained abovein the discussion of reaction with acid halides and the like.

Although the free-base form of formula I compounds can be used in themethods of the present invention, it is preferred to prepare and use apharmaceutically acceptable salt form. Thus, the compounds used in themethods of this invention primarily form pharmaceutically acceptableacid addition salts with a wide variety of organic and inorganic acids,and include the physiologically acceptable salts which are often used inpharmaceutical chemistry. Such salts are also part of this invention.Typical inorganic acids used to form such salts include hydrochloric,hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric,and the like. Salts derived from organic acids, such as aliphatic monoand dicarboxylic acids, phenyl substituted alkanoic acids,hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphaticand aromatic sulfonic acids, may also be used. Such pharmaceuticallyacceptable salts thus include acetate, phenylacetate, trifluoroacetate,acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate,naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate,caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartarate, and the like. Preferred salts are thehydrochloride and oxalate salts.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or ethyl acetate. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

The following examples are presented to further illustrate thepreparation of compounds of the present invention. It is not intendedthat the invention be limited in scope by reason of any of the followingexamples.

NMR data for the following Examples were generated on a GE 300 MHz NMRinstrument, and anhydrous d-6 DMSO was used as the solvent unlessotherwise indicated.

EXAMPLE 1 Preparation of [2-(4-methoxyphenyl)]-6-methoxynaphthyl-1-ol

6-methoxy-2-(4-methoxyphenyl)-3,4-dihydronaphthalen-1(2H)-one (8.50 g,30.14 mmol) was dissolve in 50 mL of isopropenyl acetate along with 1.0g of p-toluenesulfonic acid. The resulting mixture was heated to refluxfor 6 hours. Upon cooling to ambient temperature,2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (13.70 g, 60.28 mmol)was added. The reaction was then refluxed for 1.5 hours. After coolingto room temperature, the mixture was diluted with methylene chloride(200 mL). The organic layer was washed with 0.2N sodium hydroxide (4×200mL) followed by water (2×200 mL). The organic layer was dried (sodiumsulfate) and concentrated in vacuo to a dark solid. The crude acetatewas dissolved in 200 mL of 1:1 methanol:tetrahydrofuran and treated withexcess sodium methoxide. An orange precipitate formed that was collectedby filtration. The filtrate was acidified to pH=4 with 5N hydrochloricacid. The filtrate was further diluted with 200 mL of water and thenextracted with ethyl acetate (3×100 mL). The organic layer was dried(sodium sulfate) and concentrated in vacuo to an off-white solid.Crystallization from hexanes/ethyl acetate provided 4.24 g (50%) of[2-(4-methoxyphenyl)]-6-methoxynaphthyl-1-ol as a white solid. mp129-131° C. ¹H NMR (DMSO-d₆) δ8.19 (d, J=9.1 Hz, 1H), 7.45 (d, J=8.5 Hz,2H), 7.38 (d, J=8.0 Hz, 1H), 7.28 (d, J=8.0 Hz, 1H), 7.18 (dd, J=9.1,2.8 Hz, 1H), 7.13 (d, J=2.8 Hz, 1H), 7.07 (d, J=8.5 Hz, 2H), 5.76 (bs,1H), 3.94 (s, 3H), 3.88 (s, 3H). FD mass spec: 280. Anal. Calcd. forC₁₈H₁₆O₃: C, 77.12; H, 5.75. Found: C, 76.83; H, 5.90.

EXAMPLE 2 Preparation of1-(4-formyl)phenoxy-2-(4-methoxy-phenyl)-6-methoxynaphthalene

To a solution of [2-(4-methoxyphenyl)]-6-methoxynaphthyl-1-ol (3.57 g,12.75 mmol) in 180 mL of anhydrous N,N-dimethylformamide under N₂ atambient temperature was added sodium hydride (535 mg, 13.38 mmol, 60%dispersion in mineral oil) in small portions. After stirring for 10min., 4-flourobenzaldehyde (3.20 g, 25.50 mmol) was added. The resultingmixture was heated to 70° C. for 36 hours. Upon cooling to ambienttemperature, the solvent was removed in vacuo. The residue was thendistributed between ethyl acetate/water. The layers were separated andthe organic was washed several times with water. The organic layer wasfinally dried (sodium sulfate) and concentrated in vacuo to an oil.Chromatography (90:10 hexanes/ethyl acetate) provided 2.06 g (48%) of1-(4-formyl)phenoxy-2-(4-methoxyphenyl)-6-methoxynaphthalene as a whitesolid that was crystallized from methanol. Data for1-(4-formyl)phenoxy-2-(4-methoxyphenyl)-6-methoxynaphthalene: mp120-121° C. ¹H NMR (CDCl₃) δ9.80 (s, 1H), 7.78 (d, J=9.2 Hz, 1H), 7.74(d, J=8.8 Hz, 1H), 7.67 (d, J=8.8 Hz, 2H), 7.56 (d, J=8.8 Hz, 1H), 7.46(d, J=8.8 Hz, 2H), 7.21 (d, J=2.6 Hz, 1H), 7.12 (dd, J=9.2, 2.6 Hz, 1H),6.84 (d, J=8.8 Hz, 2H), 6.81 (d, J=8.8 Hz, 2H), 3.95 (s, 3H), 3.78 (s,3H). FD mass spec: 384. Anal. Calcd. for C₂₅H₂₀O₄: C, 78.11; H, 5.24.Found: C, 78.32; H, 5.24.

EXAMPLE 3 Preparation of1-(4-hydroxy)phenoxy-2-(4-methoxy-phenyl)-6-methoxynaphthalene

To a suspension of1-(4-formyl)phenoxy-2-(4-methoxyphenyl)-6-methoxynaphthalene (2.70 g,7.03 mmol) in 50 mL of methanol was added hydrogen peroxide (1.50 mL,14.1 mmol, 30% solution) followed by concentrated sulfuric acid (0.5mL). The reaction was gently warmed to aid dissolution then stirred atambient temperature for 36 hours. Solid sodium bicarbonate was added toneutralize the acid, and the mixture was then distributed between ethylacetate/water (100 mL ea.). The layers were separated and the organiclayer was dried (sodium sulfate) and concentrated in vacuo to a tansolid. Chromatography (silicon dioxide, 10-30% ethyl acetate/hexanes)provided 2.08 g (80%) of1-(4-hydroxy)-phenoxy-2-(4-methoxyphenyl)-6-methoxynaphthalene as awhite solid. mp 159-164° C. ¹H NMR (DMSO-d₆) δ8.91 (s, 1H). 7.76 (d,J=8.5 Hz, 1H), 7.69 (d, J=9.2 Hz, 1H), 7.54 (d, J=8.5 Hz, 1H), 7.48 (d,J=8.7 Hz, 2H), 7.37 (d, J=2.4 Hz, 1H), 7.09 (dd, J=9.2, 2.4 Hz, 1H),6.89 (d, J=8.7 Hz, 2H), 6.47 (q, J_(AB)=9.0 Hz, 4H), 3.84 (s, 3H), 3.70(s, 3H). FD mass spec: 372. Anal. Calcd. for C₂₄H₂₀O₄: C, 77.40; H,5.41. Found: C, 77.19; H, 5.70.

EXAMPLE 4 Preparation of1-[4-[2-(1-piperdinyl)ethoxy]phenoxy]-2-(4-methoxyphenyl)-6-methoxynaphthalenehydrochloride

To a solution of1-(4-hydroxy)phenoxy-2-(4-methoxy-phenyl-6-methoxynaphthalene (865 mg,2.32 mmol) in 15 mL of anhydrous N,N-dimethylformamide under N₂ wasadded cesium carbonate (3.0 g, 9.2 mmol). After stirring for 15 minutes,2-chloroethylpiperidine hydrochloride (540 mg, 2.90 mmol) was added. Theresulting mixture was stirred vigorously for 24 hours, then distributedbetween ethyl acetate/water (200 mL ea.). The layers were separated, andthe organic layer was washed several times with water. The organic layerwas then dried (sodium sulfate) and concentrated in vacuo to a whitesolid. The crude free base was dissolved in 20 mL of ethyl acetate andtreated with ethyl ether.hydrochloric acid. A precipitate formed thatwas collected by filtration and dried in vacuo to provide 1.06 g (88%)of1-[4-[2-(1-piperdinyl)ethoxy]-phenoxy]-2-(4-methoxyphenyl)-6-methoxynaphthalenehydrochloride as a white solid. mp 184-188° C. ¹H NMR (DMSO-d₆) δ9.91(bs, 1H), 7.84 (d, J=8.5 Hz, 1H), 7.71 (d, J=9.2 Hz, 1H), 7.61 (d, J=8.5Hz, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.45 (d, J=2.2 Hz, 1H), 7.15 (dd,J=9.2, 2.2 Hz, 1H), 6.93 (d, J=8.8 Hz, 2H), 6.82 (d, J=9.1 Hz, 2H), 6.61(d, J=9.1 Hz, 2H), 4.21 (m, 2H), 3.89 (s, 3H), 3.75 (s, 3H), 3.45-3.33(m, 4H), 2.92 (m, 2H), 1.80-1.63 (m, 5H), 1.35 (m, 1H). FD mass spec:483. Anal. Calcd. for C₃₁H₃₃NO₄.1.0 HCl: C, 71.60; H, 6.59; N, 2.69.Found: C, 71.77; H, 6.66; N, 2.79.

EXAMPLE 5 Preparation of1-[4-[2-(1-piperdinyl)ethoxy]phenoxy]-2-(4-hydroxyphenyl)-6-hydroxynaphthalenehydrochloride

To a solution of1-[4-[2-(1-piperdinyl)ethoxy]phenoxy]-2-(4-methoxyphenyl)-6-methoxynaphthalenehydrochloride (1.00 g, 1.92 mmol) in 20 mL of anhydrous methylenechloride under N₂ at 0° C. was added boron tribromide (0.74 mL, 7.71mmol). The resulting mixture was allowed to warm to 8° C. and stirredfor 2 hours. The reaction was then poured into a stirring solution ofcold saturated sodium bicarbonate (200 mL). When gas evolution ceased,the aqueous layer was extracted with 5% methanol/chloroform (3×100 mL).The organic layer was combined, dried (sodium sulfate), and concentratedin vacuo to an oil. The crude free base was dissolved in 20 mL of ethylacetate, and treated with ethyl ether-hydrochloric acid. A precipitateformed that was collected by filtration. Drying in vacuo provided 798 mg(83%) of1-[4-[2-(1-piperdinyl)ethoxy]-phenoxy]-2-(4-hydroxyphenyl)-6-hydroxynaphthalenehydrochloride as a white solid. mp 130-136° C. ¹H NMR (DMSO-d₆) δ9.89(s, 1H), 9.80 (bs, 1H), 9.46 (s, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.64 (d,J=9.2 Hz, 1H), 7.50 (d, J=8.5 Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.20 (d,J=2.2 Hz, 1H), 7.04 (dd, J=9.2, 2.2 Hz, 1H), 6.82 (d, J=9.1 Hz, 2H),6.76 (d, J=8.8 Hz, 2H), 6.61 (d, J=9.1 Hz, 2H), 4.21 (m, 2H), 3.45-3.33(m, 4H), 2.92 (m, 2H), 1.80-1.63 (m, 5H), 1.35 (m, 1H). FD mass spec:456. Anal. Calcd. for C₂₉H₂₉NO₄.1.0 HCl: C, 70.80; H, 6.15; N, 2.85.Found: C, 70.52; H, 6.19; N, 2.55.

Test Procedure

General Preparation Procedure

In the examples illustrating the methods, a post-menopausal model wasused in which effects of different treatments upon circulating lipidswere determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225 g) are obtained from Charles River Laboratories (Portage, Mich.).The animals are either bilaterally ovariectomized (OVX) or exposed to aSham surgical procedure at Charles River Laboratories, and then shippedafter one week. Upon arrival, they are housed in metal hanging cages ingroups of 3 or 4 per cage and have ad libitum access to food (calciumcontent approximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. Thephotoperiod in the room is 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection

After a one week acclimation period (therefore, two weeks post-OVX)daily dosing with test compound is initiated. 17α-ethynyl estradiol orthe test compound are given orally, unless otherwise stated, as asuspension in 1% carboxymethylcellulose or dissolved in 20%cyclodextrin. Animals are dosed daily for 4 days. Following the dosingregimen, animals are weighed and anesthetized with a ketamine: Xylazine(2:1, V:V) mixture and a blood sample is collected by cardiac puncture.The animals are then sacrificed by asphyxiation with CO₂, the uterus isremoved through a midline incision, and a wet uterine weight isdetermined.

Cholesterol Analysis

Blood samples are allowed to clot at ambient temperature for 2 hours,and serum is obtained following centrifugation for 10 minutes at 3000rpm. Serum cholesterol is determined using a Boehringer MannheimDiagnostics high performance cholesterol assay. Briefly, the cholesterolis oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogenperoxide is then reacted with phenol and 4-aminophenazone in thepresence of peroxidase to produce a p-quinone imine dye, which is readspectrophotometrically at 500 nm. Cholesterol concentration is thencalculated against a standard curve. The entire assay is automated usinga Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay

Uteri are kept at 4° C. until time of enzymatic analysis. The uteri arethen homogenized in 50 volumes of 50 mM Tris buffer (pH-8.0) containing0.005% Triton X-100. Upon addition of 0.01% hydrogen peroxide and 10 mMO-phenylenediamine (final concentrations) in Tris buffer, increase inabsorbance is monitored for one minute at 450 nm. The presence ofeosonophils in the uterus is an indication of estrogenic activity of acompound. The maximal velocity of a 15 second interval is determinedover the initial, linear portion of the reaction curve.

Source of Compound

17α-ethynyl estradiol was obtained from Sigma Chemical Co., St. Louis,Mo.

Osteoporosis Test Procedure

Following the General Preparation Procedure, infra, the rats are treateddaily for 35 days (6 rats per treatment group) and sacrificed by carbondioxide asphyxiation on the 36th day. The 35 day time period issufficient to allow maximal reduction in bone density, measured asdescribed herein. At the time of sacrifice, the uteri are removed,dissected free of extraneous tissue, and the fluid contents are expelledbefore determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight isroutinely reduced about 75% in response to ovariectomy. The uteri arethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

The right femurs are excised and digitilized x-rays generated andanalyzed by an image analysis program (NIH image) at the distalmetaphysis. The proximal aspect of the tibiae from these animals arealso scanned by quantitative computed tomography.

In accordance with the above procedures, compounds of the presentinvention and ethynyl estradiol (EE₂) in 20% hydroxypropylβ-cyclodextrin are orally administered to test animals.

MCF-7 Proliferation Assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.)supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM), HEPES{(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]10 mM},non-essential amino acids and bovine insulin (1 ug/mL) (maintenancemedium). Ten days prior to assay, MCF-7 cells are switched tomaintenance medium supplemented with 10% dextran coated charcoalstripped fetal bovine serum (DCC-FBS) assay medium) in place of 10% FBSto deplete internal stores of steroids. MCF-7 cells are removed frommaintenance flasks using cell dissociation medium (Ca++/Mg++ free HBSS(phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA). Cellsare washed twice with assay medium and adjusted to 80,000 cells/mL.Approximately 100 μL (8,000 cells) are added to flat-bottom microculturewells (Costar 3596) and incubated at 37° C. in a 5% CO₂ humidifiedincubator for 48 hours to allow for cell adherence and equilibrationafter transfer. Serial dilutions of drugs or DMSO as a diluent controlare prepared in assay medium and 50 μL transferred to triplicatemicrocultures followed by 50 μL assay medium for a final volume of 200μL. After an additional 48 hours at 37° C. in a 5% CO₂ humidifiedincubator, microcultures are pulsed with tritiated thymidine (1uCi/well) for 4 hours. Cultures are terminated by freezing at −70° C.for 24 hours followed by thawing and harvesting of microcultures using aSkatron Semiautomatic Cell Harvester. Samples are counted by liquidscintillation using a Wallac BetaPlace β counter. Activity of a compoundof formula I in the present assay demonstrates that the compound is ofpotential for treating hormonally-dependent cancer, particularly breastcancer.

DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Ind. Atabout 55 days of age, the rats receive a single oral feeding of 20 mg of7,12-dimethylbenz[a]anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpated at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

Compounds of Formula I are administered either through intraperitonealinjections in 2% acacia, or orally. Orally administered compounds areeither dissolved or suspended in 0.2 mL corn oil. Each treatment,including acacia and corn oil control treatments, is administered oncedaily to each test animal. Following the initial tumor measurement andselection of test animals, tumors are measured each week by theabove-mentioned method. The treatment and measurements of animalscontinue for 3 to 5 weeks at which time the final areas of the tumorsare determined. For each compound and control treatment, the change inthe mean tumor area is determined.

Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of Fibroid Tumors in Guinea Pig

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of Human Uterine Fibroid Tissue in Nude Mice

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

A. Tissue from Human Uterine Fibroid Tumors is Harvested and Maintained,in Vitro, as Primary Nontransformed Cultures

Surgical specimens are pushed through a sterile mesh or sieve, oralternately teased apart from surrounding tissue to produce a singlecell suspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, a compound of the present invention andvehicle. Levels of steroid hormone receptors are assessed weekly todetermine whether important cell characteristics are maintained invitro. Tissue from 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical Induction of Endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of Human Endometrial Tissue in Nude Mice

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

A. Tissue from Human Endometrial Lesions is Harvested and Maintained inVitro as Primary Nontransformed Cultures

Surgical specimens are pushed through a sterile mesh or sieve, oralternately teased apart from surrounding tissue to produce a singlecell suspension. Cells are maintained in media containing 10% serum andantibiotic. Rates of growth in the presence and absence of estrogen aredetermined. Cells are assayed for their ability to produce complementcomponent C3 and their response to growth factors and growth hormone. Invitro cultures are assessed for their proliferative response followingtreatment with progestins, GnRH, a compound of the invention, andvehicle. Levels of steroid hormone receptors are assessed weekly todetermine whether important cell characteristics are maintained invitro. Tissue from 5-25 patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

Inhibition of Aortal Smooth Cell Proliferation/Restenosis Test Procedure

Compounds of the present invention have capacity to inhibit aortalsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50: 172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg mlstreptomycin, 1 mC/ml ³H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95%air. At the end of 24 hours, the cells are fixed in methanol. ³Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181: 475-482(1989).

Inhibition of aortal smooth muscle cell proliferation by the compoundsof the present invention are further demonstrated by determining theireffects on exponentially growing cells. Smooth muscle cells from rabbitaortae are seeded in 12 well tissue culture plates in DMEM containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100mg/ml streptomycin. After 24 hours, the cells are attached and themedium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100U/ml penicillin, 100 mg/ml streptomycin, and desired concentrations ofthe compounds. Cells are allowed to grow for four days. Cells aretreated with trypsin and the number of cells in each culture isdetermined by counting using a ZM-Coulter counter.

Activity in the above tests indicates that the compounds of the presentinvention are of potential in the treatment of restenosis.

The present invention also provides a method of alleviatingpost-menopausal syndrome in women which comprises the aforementionedmethod using compounds of Formula I and further comprises administeringto a woman an effective amount of estrogen or progestin. Thesetreatments are particularly useful for treating osteoporosis andlowering serum cholesterol because the patient will receive the benefitsof each pharmaceutical agent while the compounds of the presentinvention would inhibit undesirable side-effects of estrogen andprogestin. Activity of these combination treatments in any of thepost-menopausal tests, infra, indicates that the combination treatmentsare useful for alleviating the symptoms of post-menopausal symptoms inwomen.

Various forms of estrogen and progestin are commercially available.Estrogen-based agents include, for example, ethynyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenichormones such as Premarin® (Wyeth-Ayerst; 0.3-2.5 mg/day).Progestin-based agents include, for example, medroxyprogesterone such asProvera® (Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), andnonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound isPremarin, and norethylnodrel and norethindrone are preferredprogestin-based agents.

The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. For the majority ofthe methods of the present invention, compounds of Formula I areadministered continuously, from 1 to 3 times daily. However, cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease. In the caseof restenosis, therapy may be limited to short (1-6 months) intervalsfollowing medical procedures such as angioplasty.

As used herein, the term “effective amount” means an amount of compoundof the present invention which is capable of alleviating the symptoms ofthe various pathological conditions herein described. The specific doseof a compound administered according to this invention will, of course,be determined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. A typical daily dose will contain a nontoxicdosage level of from about 5 mg to about 600 mg/day of a compound of thepresent invention. Preferred daily doses generally will be from about 15mg to about 80 mg/day.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Thus, another aspect of the present invention is apharmaceutical composition comprising an effective amount of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, optionallycontaining an effective amount of estrogen or progestin, and apharmaceutically acceptable carrier, diluent, or excipient.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By “pharmaceutically acceptable” itis meant the carrier, diluent, excipients and salt must be compatiblewith the other ingredients of the formulation, and not deleterious tothe recipient thereof.

Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds of formula I, with or without anestrogen or progestin compound, can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular physiological location,possibly over a period of time. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Compounds of formula I, alone or in combination with a pharmaceuticalagent of the present invention, generally will be administered in aconvenient formulation. The following formulation examples only areillustrative and are not intended to limit the scope of the presentinvention.

FORMULATIONS

In the formulations which follow, “active ingredient” means a compoundof formula I, or a salt or solvate thereof.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

Ingredient Quantity (mg/capsule) Active ingredient 0.1-1000  Starch, NF0-650 Starch flowable powder 0-650 Silicone fluid 350 centistokes 0-15 

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2: Tablets

Ingredient Quantity (mg/tablet) Active ingredient  2.5-1000 Cellulose,microcrystalline 200-650  Silicon dioxide, fumed 10-650 Stearate acid5-15

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

Formulation 3: Tablets

Ingredient Quantity (mg/tablet) Active ingredient 25-1000 Starch 45Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as 10% solutionin water) Sodium carboxymethyl cellulose 4.5 Magnesium stearate 0.5 Talc1

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 4: Suspensions

Ingredient Quantity (mg/5 ml) Active ingredient 0.1-1000 mg Sodiumcarboxymethyl cellulose 50 mg Syrup 1.25 mg Benzoic acid solution 0.10mL Flavor q.v. Color q.v. Purified water to 5 mL

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5: Aerosol

Ingredient Quantity (% by weight) Active ingredient  0.25 Ethanol 25.75Propellant 22 (Chlorodifluoromethane) 70.00

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6: Suppositories

Ingredient Quantity (mg/suppository) Active ingredient   250 Saturatedfatty acid glycerides 2,000

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 7: Intravenous Solution

Ingredient Quantity Active ingredient 50 mg Isotonic saline 1,000 mL

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

Formulation 8: Combination Capsule I

Ingredient Quantity (mg/capsule) Active ingredient 50 Premarin 1 AvicelpH 101 50 Starch 1500 117.50 Silicon Oil 2 Tween 80 0.50 Cab-O-Sil 0.25

Formulation 9: Combination Capsule II

Ingredient Quantity (mg/capsule) Active ingredient 50 Norethylnodrel 5Avicel pH 101 82.50 Starch 1500 90 Silicon Oil 2 Tween 80 0.50

Formulation 10: Combination Tablet

Ingredient Quantity (mg/capsule) Active ingredient 50 Premarin 1 CornStarch NF 50 Povidone, K29-32 6 Avicel pH 101 41.50 Avicel pH 102 136.50Crospovidone XL10 2.50 Magnesium Stearate 0.50 Cab-O-Sil 0.50

I claim:
 1. A method for treating pathological conditions resulting froman estrogen deficiency comprising administering to a women in need oftreatment an effective amount of a compound of formula I

wherein R¹ is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), or—OSO₂(C₂-C₆ alkyl); R² is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆alkyl), —OSO₂(C₂-C₆ alkyl), or halo; R³ is 1-piperidinyl,1-pyrrolidinyl, methyl-1-pyrrolidinyl, dimethyl-1-pyrrolidino,4-morpholino, dimethylamino, diethylamino, diisopropylamino, or1-hexamethyleneimino; and n is 2 or 3; and or a pharmaceuticallyacceptable salt thereof.
 2. A method according to claim 1 wherein thepathological condition is osteoporosis.
 3. A method according to claim 1wherein the pathological condition is related to a cardiovasculardisease.
 4. A method according to claim 3 wherein the cardiovasculardisease is hyperlipidemia.
 5. A method for treating estrogen-dependentcancer comprising administering to a woman in need of treatment aneffective amount of a compound of formula I

wherein R¹ is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), or—OSO₂(C₂-C₆ alkyl); R² is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆alkyl), —OSO₂(C₂-C₆ alkyl), or halo; R³ is 1-piperidinyl,1-pyrrolidinyl, methyl-1-pyrrolidinyl, dimethyl-1-pyrrolidino,4-morpholino, dimethylamino, diethylamino, diisopropylamino, or1-hexamethyleneimino; and n is 2 or 3; or a pharmaceutically acceptablesalt thereof.
 6. A method according to claim 5 wherein theestrogen-dependent cancer is breast or uterine cancer.
 7. A methodaccording to claim 6 wherein R¹ and R² of said formula I compound eachare —OH, R³ is 1-piperidinyl, and n is
 2. 8. A method according to claim7 wherein said salt thereof is the hydrochloride salt.
 9. A method forcomprising treating pathological conditions resulting from an estrogendeficiency administering to a woman in need of treatment an effectiveamount of a compound of formula I

or a pharmaceutically acceptable salt thereof wherein R¹ is —H, —OH,—O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), or —OSO₂(C₂-C₆ alkyl); R²is —H, —OH, —O(C₁-C₄ alkyl), —OCOC₆H₅, —OCO(C₁-C₆ alkyl), —OSO₂(C₂-C₆alkyl) or halo; R³ is 1-piperidinyl, 1-pyrrolidinyl,methyl-1-pyrrolidinyl, dimethyl-1-pyrrolidinyl, 4-morpholino,dimethylamino, diethylamino, diisopropylamino, or 1-hexamethyleneimino;and n is 2 or 3; in combination with an effective amount of estrogen.